Oligodendroglioma drive genes

ABSTRACT

Oligodendrogliomas are the second most common malignant brain tumor in adults. These tumors often contain a chromosomal abnormality involving a pericentromeric fusion of chromosomes 1 and 19, resulting in losses of the entire short arm of the former and the long arm of the latter. To identify the molecular genetic basis for this alteration, we performed exomic sequencing of seven anaplastic oligodendrogliomas with chromosome 1p and 19q losses. Among other changes, we found that that CIC (homolog of the  Drosophila  gene capicua) on chromosome 19q was somatically mutated in six of the seven cases and that FUBP1 (far upstream element (FUSE) binding protein) on chromosome 1p was somatically mutated in two of the seven cases. Examination of 27 additional oligodendrogliomas revealed 12 and 3 more tumors with mutations of CIC and FUBP1, respectively, 58% of which were predicted to result in truncations of the encoded proteins. These results suggest a critical role for these genes in the biology and pathology of oligodendrocytes.

STATEMENT OF FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant No. CA43460,awarded by the National Institutes of Health. The government has certainrights in the invention.

TECHNICAL FIELD OF THE INVENTION

This invention is related to the area of cancer. In particular, itrelates to brain cancers.

BACKGROUND OF THE INVENTION

Oligodendrogliomas (ODs) account for 20% of brain tumors in adults and,as their name suggests, they have prominent oligodendroglialdifferentiation (1, 2). These tumors generally arise in the white matterof cerebral hemispheres, in the frontal lobes. Well-differentiated ODscan evolve into high-grade “anaplastic” ODs, though it is oftendifficult to clearly distinguish these two types from each other or fromother brain tumors (1, 2). Because this distinction is important for themanagement of patients, molecular biomarkers for ODs are of greatinterest.

To date, the best biomarker for ODs is loss of heterozygosity (LOH) ofchromosomes 1p and 19q (2-5). Assessment for LOH events is now commonlyperformed in patients with ODs because of their important implicationsfor therapeutic responses (2-5). The chromosome losses occur in 50% to70% of tumors and are often associated with a pericentromerictranslocation of chromosomes 1 and 19, producing marker chromosomeder(1;19) (q10;p10) (2-7). This translocation is unbalanced, leaving thecells with one copy of the short arm of chromosome 1 and one copy of thelong arm of chromosome 19. The functional basis for most cancertranslocations involves one of the genes residing near the breakpoints,producing fusions that alter the gene's product. In contrast, theder(1;19) (q10;p10) breakpoints are in gene-poor centromeric regions andare always associated with LOH (4, 6, 8). This suggests that the basisfor the t(1;19) translocation is the unmasking of a tumor suppressorgene(s) on either chromosome 1p or 19q (2-5), (9). This is supported bythe fact that some tumors lose only chromosome 1p sequences, whileothers lose only chromosome 19q sequences.

There is a continuing need in the art to identify this putative tumorsuppressor gene(s), as well as to increase understanding of ODpathogenesis.

SUMMARY OF THE INVENTION

One aspect of the invention is a method of identifying anoligodendroglioma. A sample is tested for an inactivating mutation inCIC, FUBP1, or both CC and FUBP. The sample is from a brain tissuesuspected of being a brain tumor, or in cells or nucleic acids shed fromthe tumor. The presence of the inactivating mutation indicates anoligodendroglioma.

Another aspect of the invention is a method of stratifying a patientwith a brain tumor. A sample is tested for an inactivating mutation ofCIC. The sample is from a brain tumor or cells or nucleic acids shedfrom the tumor. Brain tumors with the mutation are refractory to EGFRinhibitors.

Yet another aspect of the invention is a method of predicting survivalfor a patient with an oligodendroglioma. A sample is tested for aninactivating mutation in CIC, FUBP1 or both CIC and FUBP1. The sample isfrom the oligodendroglioma or cells or nucleic acids shed from theoligodendroglioma. Presence of the mutation portends an improvedsurvival relative to oligodendroglioma patients without the inactivationmutation. Absence of the mutation portends a decreased survival relativeto oligodendroglioma patients with the inactivation mutation.

Still another aspect of the invention is a method of predictingchemotherapy response or radiotherapy response of an oligodendroglioma.A sample is tested for an inactivating mutation in CIC, FUBP1 or bothCIC and FUBP1 in the oligodendroglioma. Presence of the inactivatingmutation portends a positive response to chemotherapy or radiotherapy.Absence of the inactivating mutation portends a negative response tochemotherapy or radiotherapy.

One aspect of the invention is a method of monitoring status of apatient with a brain tumor that has an inactivating mutation in CIC,FUBP1 or both CIC and FUBP1. A sample of blood or cerebrospinal fluidfrom the patient is tested to determine an amount of nucleic acids withan inactivating mutation in CIC, FUBP1 or both CIC and FUBP. The step oftesting is repeated one or more times with samples taken at distincttime points. An increase in the amount of the nucleic acids indicates anincrease in the amount of brain tumor. A decrease in the amount of thenucleic acids indicates a decrease in the amount of brain tumor. Anequivalent amount of the nucleic acids indicates an equivalent amount ofbrain tumor.

These and other embodiments which will be apparent to those of skill inthe art upon reading the specification provide the art with tools fordiagnosis, prognosis, treatment, and assessment of brain cancers.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-FIG. 1B. Loss of heterozygosity (LOH) maps of two representativetumors. (FIG. 1A) In tumor OLID 13, the estimated LOH on chromosome 1extends from base 901,779 to base 148,526,024 and the estimated LOH onchromosome 19 extends from base 18,116,940 to base 62,357,562. (FIG. 1B)In tumor OLID 09, the estimated LOH on chromosome 1 extends from base1,844,406 to base 110,751,800, the estimated LOH on chromosome 9 extendsfrom base 108,032 to base 20,875,240 and the estimated LOH on chromosome19 extends from base 18,545,563 to base 62,923,619. The “minor allele”of each SNP represents the allele that was less common in the tumor. Ifboth alleles of the SNP were represented by an equal number of tags, theminor allele fraction would be represented as 100% on the y-axis. Theremaining signals in the regions exhibiting LOH represent contaminatingnon-neoplastic cells in the samples.

FIG. 2A-FIG. 2B Mutations in CIC and FUBP1. (FIG. 2A) Sanger sequencingchromatograms showing representative CIC or FUBP1 mutations in theindicated tumors. T, DNA from tumor; N, DNA from matched normal tissue.The mutated bases are overlined with a red bar. (FIG. 2B) Mutationdistribution of CIC mutations. Red arrows represent missense mutationssubstitutions, black arrows represent insertions or deletions, and greenarrows represent splice site alterations. See Tables s2 and s3 fordetails. The black boxes denote exons, Pro-rich denotes the proline-richdomains, HMG denotes the high mobility group domain, and the start andstop codons are indicated.

FIG. 3A-3E (Fig. s1) Loss of heterozygosity (LOH) maps of the remainingdiscovery screen samples. (FIG. 3A) In tumor OLID 2, the estimated LOHon chromosome 1 extends from base 1,640,705 to base 113,038,204, onchromosome 4 from base 41,310,447 to base 187,775,127, on chromosome 9from base 10,47,204 to base 17,263,878, on chromosome 13 from base24,254,053 to base 102,272,383, on chromosome 15 from base 27,202,852 tobase 89,313,271, on chromosome 18 from base 6,975,631 to base 58,781,511and the estimated LOH on chromosome 19 extends from base 18,835,200 tobase 63,681,236. (FIG. 3B) In tumor OLID 8, the estimated LOH onchromosome 1 extends from base 1,640,705 to base 112,100,105, onchromosome 4 from base 1,077,531 to base 186,509,767, on chromosome 9from base 123,968,827 to base 138,453,090 and the estimated LOH onchromosome 19 extends from base 17,525,418 to base 62,340,089. (FIG. 3C)In tumor OLID 15, the estimated LOH on chromosome 1 extends from base1,129,725 to base 111,695,481, on chromosome 9 from base 115,970,015 tobase 139,897,127 and the estimated LOH on chromosome 19 extends frombase 17,589,502 to base 61,645,775. (FIG. 3D) In tumor OLID 10, theestimated LOH on chromosome 1 extends from base 1,640,705 to base120,413,529, on chromosome 9 from base 108,032 to base 138,820,929 andthe estimated LOH on chromosome 19 extends from base 18,146,944 to base62,601,639. (FIG. 3E) In tumor OLID 12, the estimated LOH on chromosome1 extends from base 939,471 to base 111,767,814, on chromosome 4 frombase 3,008,948 to base 186,509,767, on chromosome 9 from base 2,707,698to base 38,005,327, on chromosome 15 from base 22,879,808 to base97,285,457 and the estimated LOH on chromosome 19 extends from base22,048,143 to base 63,560,292.

FIG. 4 (Table s1) Clinical characteristics of the patients and tumors

FIG. 5 (Table s2) Somatic mutations in oligodendrogliomas

FIG. 6 (Table s3) Mutations identified in the validation samples

DETAILED DESCRIPTION OF THE INVENTION

The inventors have developed methods for identifying, stratifying,prognosing, theranosing, and monitoring brain tumors, particularlyoligodendrogliomas. The methods center around two genes which were foundto be frequently mutated in such brain tumors, CIC and FUBP1. Mutationsof many types have been found. The spectrum of mutations indicates thatthe mutations inactivate the gene products, identifying the genes astumor suppressors.

Tests for CIC and FUBP1 mutations can be performed using protein basedor nucleic based assays. Sequence determination of the nucleic acid canbe used to identify mutations. Probes or primers, and kits andtechniques employing both can be used. PCR or other specific or globalamplification can be used. Mutations can be identified in any availablegenetic material including, for example, genomic DNA, cDNA, and RNA.Nucleic acids can be amplified, enriched, and/or purified prior toassessment. Protein based assays may involve specific antibodies and/orCIC and FUBP1 binding partners such as PUF60. The antibodies may bepolyclonal or monoclonal, fragments (Fab, Fab′), single chain constructs(scFv), etc. Nucleic acid based assays include without limitation,hybridization to probes, amplification using specific primers, primerextension, ligation assay, etc. Any of these techniques can also becombined. Assays can be performed together with tests for other genemutations or alterations of the genome. Results can be integrated andused to accurately and comprehensively characterize and/or identify atumor or the patient.

Results of assays can be recorded in a written medium, an electronicmedium, or transmitted orally or electronically to a health careprovider, a patient, a family member, a hospital, a medical record, etc.Testing requires physical steps, and typically involves chemical changesto occur to a test sample. Typically the test sample is a sample that isremoved from the patient body, so that the test is performed outside ofa patient body.

Samples which may be tested include without limitation brain tissue,tumor tissue, CNS fluid, neuronal tissue, blood, urine, saliva, tears,sputum, etc. These samples may be collected and processed and/or storedprior to testing. For example, serum or plasma samples derived fromblood may be used in an assay. The samples may be frozen or fixed. Theymay be archival or freshly collected.

Any type of mutation may be identified. Inactivating mutations includewithout limitation CIC mutations in the genome g.chr19:47483555C>T;g.chr19:47483592G>C; g.chr19:47483598G>A; g.chr19:47486574delGT;g.chr19:47487549G>A; g.chr19:47490688G>T; g.chr19:47485924insG;g.chr19:47490903delAGA; g.chr19:47483711G>A; g.chr19:47490722C>T;g.chr19:47483597C>T; g.chr19:47483438delC; g.chr19:47483952G>A;g.chr19:47490203delCGCAAGATGAGAAGACG (SEQ ID NO: 1); andg.chr19:47490728G>GC; CIC mutations in cDNA c.601C>T; c.638G>C;c.644G>A; c.1814delGT; IVS10-1G>A; c.4420G>T; c.1445insG; c.4547delAGA;c.757G>A; c.4454C>T; c.643C>T; c.579delC; c.916G>A;c.4234delCGCAAGATGAGAAGACG (SEQ ID NO: 1); c.643C>T; and IVS4459+1. Themutation may be a frameshift mutation, a splice-site mutation, an indel(insertion or deletion) mutation, or a missense mutation. Particularmutations which may be identified include p.R201W; p.R213P; p.R215Q;p.QK1517RD; p.A253T; p.P1485L; p.A306T; p.R215W; and p.V1474F.

Inactivating mutations in FUBP1 include without limitation genomicmutations chr1:78201054G>T; chr1:78206439delACTG; chr1:78193600delG;g.chr1:78198726delG; g.chr1:78201156C>A, and mutations in the cDNA atc.1333G>T; c.248delACTG; c.1538delC; c.1231G>T; c.1708delG. The mutationmay be a nonsense, deletion, or frameshift, for example. Particularmutations include p.E445X and p.E411X.

Stratification of patients can be used to assign a treatment regimen. Itmay be used in prospective or retrospective clinical studies. It can beused to assign a prognosis or a prediction regarding survival orchemotherapy or radiotherapy sensitivity. Stratification typicallyassigns a patient to a group based on a shared mutation pattern or otherobserved characteristic or set of characteristics.

Predictions of survival can be based on one or more characteristic of adisease or patient having the disease. Predictions based on onecharacteristic can be modified by other characteristics, making thepredictions more accurate. The characteristic inactivating mutations inCIC and FUBP1 can be used individually or in combination with each otheror with other characteristics. Predictions of survival rates or timescan be communicated and/or recorded for the patient, other health careprofessionals, the medical record of the patient, the patient's family,etc. Such predictions are typically made by comparing survival data fora group of patients that share one or more characteristics with thepatient.

The mutations in CIC and FUBP1 can be similarly used to design atreatment plan. The treatment plan can take into consideration whichdrugs or other therapies are typically effective in tumors with thesemutations and which drugs or other therapies are typically ineffective.Thus the mutation status can be used to make a decision to treat or adecision not to treat with a particular agent.

A brain tumor, such as an oligodendroglioma, can be monitored over timeusing the nucleic acids with the CIC and FUBP1 mutations as a marker ofthe tumor. The monitoring can be used as a means to detect recurrence,or growth and progression of an existing tumor. The monitoring can beused to measure response to a therapeutic regimen. At least two timepoints are assessed so that changes over time can be determined. Anysuitable control sample can be used for means of normalizing results.These may include a non-cancer specific nucleic acid marker, such as ahousekeeping gene, or wild-type versions of the CIC and FUBP1 genes, orthe total amount of nucleic acids. Those of skill in the art willrecognize best ways to normalize the data.

The capicua gene was discovered in a screen for mutations affecting theanteroposterior pattern of Drosophila embryos (29). Females withinactivating CIC mutations produce embryos that form head and tailstructures but lack most intervening segments (capicua means“head-and-tail” in Catalan). In Drosophila, the protein encoded by CIChas been shown to be a downstream component of receptor tyrosine kinase(RTK) pathways that includes EGFR, Torso, Ras, Raf, andmitogen-associated protein kinases (MAPKs) (30, 31). In the absence ofRTK signaling, cic, in combination with other transcription factors suchas Groucho (Gro), blocks transcription by binding to canonical octamericelements in regulatory regions (32). RTK signaling blocks the functionof cic via MAPK-mediated phosphorylation or docking, resulting indegradation of cic and the consequent activation of the genes itnormally represses (33). The most highly conserved functional domain ofthe cic protein is the HMG (high mobility group) box responsible for itsbinding to DNA. Importantly, 8 of the 11 missense mutations we observedin ODs were located in this domain (FIG. 2B).

In addition to the high conservation of CIC sequences among metazoans,the human cic protein contains nine consensus phosphorylation sites forMAPK(34). This suggests that human cic functions similarly to itsDrosophila counterpart. This hypothesis is supported by massspectroscopic studies that have shown human cic protein to bephosphorylated within 10 minutes of EGF treatment of HeLa cells (35).Genetic alterations of EGFR are common in glioblastomas (36, 37),prompting clinical trials of EGFR inhibitors (38). However, epistaticexperiments in Drosophila (31) show that that cic is downstream of EGFR,suggesting that EGFR inhibitors would not be useful in ODs with CICmutations.

The protein encoded by FUBP1 binds to single stranded DNA, in particularthe far-upstream element (FUSE) of MYC, a well-studied oncogene (39).Although overexpression of FUBP1 can stimulate MYC expression (39), ithas also been shown that FUBP1 protein participates in a complex withPUF60 that negatively regulates MYC expression (40). Our data, showingthat FUBP1 is inactivated by mutations, are consistent with the ideathat FUBP1 mutations lead to MYC activation in these tumors by relievingthe negative effects of the FUBP1-PUF60-FUSE complex.

There are only a small and statistically insignificant number of pointmutations of FUBP1 or CIC recorded in the COSMIC database (41). However.CIC has shown to be translocated in two cases of Ewing's sarcoma-liketumors that harbored t(4;19)(q35;q13) translocations. Unlike themutations observed in ODs, the translocations in these two cases seemedto activate the cic protein by fusing it to the C-terminus of DUX4,conferring oncogenic properties to the new protein (42).

Overall, 23 mutations of CIC or FUBP1 were identified in the 34 tumorsanalyzed in this study. As our mutational screens would not detect sometypes of inactivating mutations (e.g., large deletions or promotermutations) or epigenetic alterations, the fraction of tumors withdetectable CIC and FUBP1 mutations is likely an underestimate of theiractual contribution.

How do the der(1;19) (q10;p10) chromosomes arise? One possibility isthat the pericentromeric translocation of chromosomes 1 and 19 is facileway to inactivate CIC given the high homology between the centromeres ofthese two chromosomes (43). In this scenario, the unbalancedtranslocation event would be solely driven by CIC inactivation.Inactivation of tumor suppressor genes on 1p, such as FUBP1, NOTCH2,MAP3KC, and CDKN2C (FIG. 5; Table s2) would then represent opportunisticevents in a subset of the tumors with CIC mutations. However, the factthat two of five ODs with FUBP1 mutations did not have detectable CICmutations argues against this model. The converse situation, in whichthe initial driver event is an inactivation of FUBP1, subsequentlyfollowed in some cases by mutational inactivation of CIC, is thereforealso possible. These scenarios are consistent with the demonstrationthat losses of chromosome 1p do not always occur in conjunction withlosses of chromosome 19, and vice versa (2-5). Regardless of the chainof events, our identification of inactivating mutations of CIC or FUBP1in a substantial fraction of ODs are likely to provide importantinsights into the pathogenesis of these tumors as well as help refinetheir diagnosis, prognosis, and treatment options.

The above disclosure generally describes the present invention. Allreferences disclosed herein are expressly incorporated by reference. Amore complete understanding can be obtained by reference to thefollowing specific examples which are provided herein for purposes ofillustration only, and are not intended to limit the scope of theinvention.

Example 1

We sequenced the coding exons of 20,687 genes in DNA from sevenanaplastic ODs and compared them to the sequences of DNA from normalleukocytes of the same patients. All seven tumors had been shown to haveLOH of chromosome 1p and 19q using approved clinical assays. Theclinical characteristics of the patients and their tumors are listed inFIG. 4; Table s1. DNA from enriched neoplastic cells and matched normalcells was sheared and used to prepare fragment libraries suitable formassively parallel sequencing (10). The coding sequences of the targetedgenes were captured with the 50 MB SureSelect Enrichment System andsequenced using the Illumina HiSeq platform. The average coverage ofeach base in the targeted regions was high (135-fold), and 94% of thebases were represented by at least 10 reads (Table 1).

Table 1A and 1B: Summary of Sequence Analysis of Oligodendrogliomas

TABLE 1A Coverage Summary Tumor Normal Bases sequenced (after 12.3 ± 4.0× 109 11.8 ± 1.3 × 109 quality filtering) Bases mapped to targeted 7.36± 2.3 × 109  7.32 ± 0.82 × 109 region Average # of reads per 135 ±42.2    135 ± 12.2    targeted base Targeted bases with at 94 ± 1.1%  95± 0.5%  least 10 reads (%)

TABLE 1B Tumor and normal comparison Known SNPs identified in tumor22,817 ± 1083  % tumor SNPs identified in matched normal  99.7 ± 0.02%Non-synonymous somatic mutations in 32.1 ± 10.7 tumor

As with complete genomic sequencing (11, 12), exomic sequencing canidentify chromosomal regions that undergo loss of heterozygosity (LOH)using common single nucleotide polymorphisms (SNPs) identified to beheterozygous in DNA from corresponding normal cells. There were14,032±540 SNPs per patient that could be used for this analysis. Anexample is provided in FIG. 1A, indicating that the only regionsexhibiting LOH in tumor OLID 13 were on chromosomes 1p and 19q. Anotherexample is in FIG. 1B, showing that tumor OLID 09 had lost loci onchromosomes 9p as well as 1p and 19q. LOH on chromosome 9p occurs in athird of ODs and likely reflects inactivation of the CDKN2A tumorsuppressor gene (13). All seven ODs analyzed by genomic sequencingexhibited LOH of alleles spanning the entire short arm of chromosome 1and the entire long arm of chromosome 19 (FIG. 3 (s1)). Other recurrentchanges were on chromosome 9p (four tumors), 4q (three tumors), and 15q(two tumors) (FIG. 3 (s1)).

Example 2

We have previously described methods for the accurate identification ofsomatic mutations in next-generation sequencing data from Illuminainstruments (14, 15). Using these stringent criteria to avoid falsepositive calls, we identified a total of 225 non-synonymous somaticmutations, affecting 200 genes, among the seven tumors (FIG. 5; FIG. 5;Table s2). There were an average of 32.1±10.7 non-synonymous somaticmutations per tumor (Table 1), similar to the number found in the mostcommon type of adult brain tumor (glioblastoma, 35.6 non-synonymoussomatic mutations per tumor (16)).

There were a number of notable mutations identified in these seventumors. Three tumors with mutations in PIK3CA were identified, eachoccurring in a previously defined “hotspot” for recurrent mutations inother tumor types (FIG. 5; Table s2) (17). PIK3CA encodes the catalyticsubunit of the PI3Kα lipid kinase (18-20). A fourth tumor had a 3-basepair deletion in PIK3R1, the gene encoding the regulatory subunit of thePI3Kα enzyme; in-frame deletions of PIK3R1 are relatively common inother types of brain tumors (16) (21), and are likely to enhance theactivity of the catalytic subunit (18-20). The NOTCH1 gene was mutatedin two tumors and at least one of these was inactivating (a 1 bpdeletion), consistent with the recently described tumor suppressor rolefor this gene (22). Finally, the IDH1 (isocitrate dehydrogenase 1) genewas mutated in all seven tumors at the same residue, resulting in anamino acid substitution of His for Arg at codon 132. A high frequency ofIDH1 mutations in ODs has been previously documented (23), (24) andshown to produce neo-enzymatic activity resulting in the abnormalproduction of 2-hydroxyglutarate (25).

Example 3

One of the major goals of this study was the investigation of the targetgene(s) on chromosome 1 or 19. By analogy with other tumor suppressorgenes (26), (27) we expected that the residual copy of the targetgene(s) would contain mutations in most tumors with LOH of the relevantregion. On chromosome 1p, there were eight somatically mutated genes,but only two with mutations in more than one tumor: FUBP1 (Far UpstreamElement [FUSE] Binding Protein 1) and NOTCH2 (FIG. 5; Table s2). Onchromosome 19q, there were three genetically altered genes identified,two of which were mutated in a single tumor each. The third, CIC(homolog of the Drosophila capicua gene), was mutated in six of theseven tumors. In each of these six cases, the fraction of mutant alleleswas high (80.5±10.7%), consistent with loss of the non-mutated allele.The mutations were confirmed to be homozygous by Sanger sequencing (FIG.2A).

Example 4

To validate these results and determine the spectrum of FUBP1, NOTCH2,and CIC mutations in ODs, we examined tumor DNA from an additional 27tumors and matched normal cells. No additional mutations of NOTCH2 werefound, but FUBP1 and CIC mutations were identified in 3 and 12 of theadditional cases and generally (14 of 16 mutations) appeared to behomozygous (FIG. 2B, FIG. 6; Table s3). The probability that thesemutations were passengers rather than drivers was <10⁻⁸ for both genes(binomial test, (28). All FUBP1 mutations and more than 25% of the CICmutations were predicted to inactivate their encoded proteins, as theyaltered splice sites, produced stop codons, or generated out-of-frameinsertions or deletions (FIG. 2B and FIG. 6; Table s3). This type ofmutational pattern is routinely observed in tumor suppressor genes suchas TP53 or FBXW7 (41) but is never observed in bona fide oncogenes.

Example 5

Materials and Methods

Preparation of Illumina Genomic DNA Libraries

Fresh-frozen surgically resected tumor and matched blood were obtainedfrom patients under an Institutional Review Board protocol. Tumor tissuewas analyzed by frozen section histology to estimate neoplasticcellularity. Genomic DNA libraries were prepared following Illumina's(Illumina, San Diego, Calif.) suggested protocol with the followingmodifications. (1) 1-3 micrograms (μg) of genomic DNA from tumor orlymphocytes in 100 microliters (μl) of TE was fragmented in a Covarissonicator (Covaris, Woburn, Mass.) to a size of 100-500 bp. To removefragments shorter than 150 bp, DNA was mixed with 25 μl of 5× Phusion HFbuffer, 416 μl of ddH2O, and 84 μl of NT binding buffer and loaded intoNucleoSpin column (cat #636972. Clontech, Mountain View, Calif.). Thecolumn was centrifuged at 14000 g in a desktop centrifuge for 1 min,washed once with 600 μl of wash buffer (NT3 from Clontech), andcentrifuged again for 2 min to dry completely. DNA was eluted in 45 μlof elution buffer included in the kit. (2) Purified, fragmented DNA wasmixed with 40 μl of H2O, 10 μl of End Repair Reaction Buffer, 5 μl ofEnd Repair Enzyme Mix (cat # E6050, NEB, Ipswich, Mass.). The 100 μlend-repair mixture was incubated at 20° C. for 30 min, purified by a PCRpurification kit (Cat #28104, Qiagen) and eluted with 42 μl of elutionbuffer (EB). (3) To A-tail, all 42 μl of end-repaired DNA was mixed with5 μl of 10×dA Tailing Reaction Buffer and 3 μl of Klenow (exo-) (cat #E6053, NEB, Ipswich, Mass.). The 50 μl mixture was incubated at 37° C.for 30 min before DNA was purified with a MinElute PCR purification kit(Cat #28004, Qiagen). Purified DNA was eluted with 25 μl of 70° C. EB.(4) For adaptor ligation, 25 μl of A-tailed DNA was mixed with 10 μl ofPE-adaptor (Illumina), 10 μl of 5× Ligation buffer and 5 μl of Quick T4DNA ligase (cat # E6056, NEB, Ipswich, Mass.). The ligation mixture wasincubated at 20° C. for 15 min. (5) To purify adaptor-ligated DNA, 50 μlof ligation mixture from step (4) was mixed with 200 μl of NT buffer andcleaned up by NucleoSpin column. DNA was eluted in 50 μl elution buffer.(6) To obtain an amplified library, ten PCRs of 50 μl each were set up,each including 29 μl of H2O, 10 μl of 5× Phusion HF buffer, 1 μl of adNTP mix containing 10 mM of each dNTP, 2.5 μl of DMSO, 1 μl of IlluminaPE primer #1, 1 μl of Illumina PE primer #2, 0.5 μl of Hotstart Phusionpolymerase, and 5 μl of the DNA from step (5). The PCR program used was:98° C. 2 minute; 6 cycles of 98° C. for 15 seconds, 65° C. for 30seconds, 72° C. for 30 seconds; and 72° C. for 5 min. To purify the PCRproduct, 500 μl PCR mixture (from the ten PCR reactions) was mixed with1000 μl NT buffer from a NucleoSpin Extract II kit and purified asdescribed in step (1). Library DNA was eluted with 70° C. elution bufferand the DNA concentration was estimated by absorption at 260 nm.

Exome and Targeted Subgenomic DNA Capture

Human exome capture was performed following a protocol from Agilent'sSureSelect Paired-End Target Enrichment System (All Exon 50 Mb kit,Agilent, Santa Clara, Calif.) with the following modifications. (1) Ahybridization mixture was prepared containing 25 μl of SureSelect Hyb#1, 1 μl of SureSelect Hyb #2, 10 μl of SureSelect Hyb #3, and 13 μl ofSureSelect Hyb #4. (2) 3.4 μl (0.5 μg) of the PE-library DNA describedabove, 2.5 μl of SureSelect Block #1, 2.5 μl of SureSelect Block #2 and0.6 μl of Block #3; was loaded into one well in a 384-well Diamond PCRplate (cat # AB-1111, Thermo-Scientific, Lafayette, Colo.), sealed withmicroAmp clear adhesive film (cat #4306311; ABI, Carlsbad, Calif.) andplaced in GeneAmp PCR system 9700 thermocycler (Life Sciences Inc.,Carlsbad Calif.) for 5 minutes at 95° C., then held at 65° C. (with theheated lid on). (3) 25-30 μl of hybridization buffer from step (1) washeated for at least 5 minutes at 65° C. in another sealed plate withheated lid on. (4) 5 μl of SureSelect Oligo Capture Library, 1 μl ofnuclease-free water, and 1 μl of diluted RNase Block (prepared bydiluting RNase Block 1:1 with nuclease-free water) were mixed and heatedat 65° C. for 2 minutes in another sealed 384-well plate. (5) Whilekeeping all reactions at 65° C., 13 μl of Hybridization Buffer from Step(3) was added to the 7 μl of the SureSelect Capture Library Mix fromStep (4) and then the entire contents (9 μl) of the library from Step(2). The mixture was slowly pipetted up and down 8 to 10 times. (6) The384-well plate was scaled tightly and the hybridization mixture wasincubated for 24 hours at 65° C. with a heated lid.

After hybridization, five steps were performed to recover and amplifycaptured DNA library: (1) Magnetic beads for recovering captured DNA: 50μl of Dynal MyOne Streptavidin C1 magnetic beads (Cat #650.02,Invitrogen Dynal, AS Oslo, Norway) was placed in a 1.5 ml microfuge tubeand vigorously resuspended on a vortex mixer. Beads were washed threetimes by adding 200 μl of SureSelect Binding buffer, mixed on a vortexfor five seconds, and placed in a Dynal magnetic separator to remove thesupernatant. After the third wash, beads were resuspended in 200 μl ofSureSelect Binding buffer. (2) To bind captured DNA, the entirehybridization mixture described above (29 μl) was transferred directlyfrom the thermocycler to the bead solution and mixed gently; thehybridization mix/bead solution was incubated in an Eppendorfthermomixer at 850 rpm for 30 minutes at room temperature. (3) To washthe beads, the supernatant was removed from the beads after applying aDynal magnetic separator and the beads were resuspended in 500 μlSureSelect Wash Buffer #1 by mixing on a vortex mixer for 5 seconds andincubated for 15 minutes at room temperature. Wash Buffer #1 was thenremoved from the beads after magnetic separation. The beads were furtherwashed three times, each with 500 μl pre-warmed SureSelect Wash Buffer#2 after incubation at 65° C. for 10 minutes. After the final wash,SureSelect Wash Buffer #2 was completely removed. (4) To elute capturedDNA, the beads were suspended in 50 μl SureSelect Elution Buffer,vortex-mixed and incubated for 10 minutes at room temperature. Thesupernatant was removed after magnetic separation, collected in a new1.5 ml microcentrifuge tube, and mixed with 50 μl of SureSelectNeutralization Buffer. DNA was purified with a Qiagen MinElute columnand eluted in 17 μl of 70° C. EB to obtain 15 μl of captured DNAlibrary. (5) The captured DNA library was amplified in the followingway: 15 PCR reactions each containing 9.5 μl of H2O, 3 μl of 5× PhusionHF buffer, 0.3 μl of 10 mM dNTP, 0.75 μl of DMSO, 0.15 μl of Illumina PEprimer #1, 0.15 μl of Illumina PE primer #2, 0.15 μl of Hotstart Phusionpolymerase, and 1 μl of captured exome library were set up. The PCRprogram used was: 98° C. for 30 seconds; 14 cycles of 98° C. for 10seconds, 65° C. for 30 seconds, 72° C. for 30 seconds; and 72° C. for 5min. To purify PCR products, 225 μl PCR mixture (from 15 PCR reactions)was mixed with 450 μl NT buffer from NucleoSpin Extract II kit andpurified as described above. The final library DNA was eluted with 30 μlof 70° C. elution buffer and DNA concentration was estimated by OD260measurement.

Somatic Mutation Identification by Massively Parallel Sequencing

Captured DNA libraries were sequenced with the Illumina GAIIx/HiSeqGenome Analyzer, yielding 150 (2×75) base pairs from the final libraryfragments. Sequencing reads were analyzed and aligned to human genomehg18 with the Eland algorithm in CASAVA 1.7 software (Illumina). Amismatched base was identified as a mutation only when (i) it wasidentified by more than five distinct tags; (ii) the number of distincttags containing a particular mismatched base was at least 20% of thetotal distinct tags; and (iii) it was not present in >0.1% of the tagsin the matched normal sample. SNP search databases included that of theNational Library Of Medicine and that of 1000 Genomes.

Evaluation of Genes in Additional Tumors and Matched Normal Controls.

The somatic mutations in CIC, FUBP1, and NOTCH2 in the Discovery setwere confirmed by Sanger sequencing as described previously (1). Theentire coding regions of CIC, FUBP1, and NOTCH2 were sequenced in avalidation set composed of an independent series of additionaloligodendrogliomas and matched controls. PCR amplification and Sangersequencing were performed as described in T. Sjoblom et al., Science,268 (2006).

REFERENCES

The disclosure of each reference cited is expressly incorporated herein.

-   1. S. W. Coons, P. C. Johnson, B. W. Scheithauer, A. J. Yates, D. K.    Pearl, Cancer 79, 1381 (1997).-   2. J. E. Bromberg, M. J. van den Bent, Oncologist 14, 155 (2009).-   3. D. Maintz et al., J Neuropathol Exp Neurol 56, 1098 (1997).-   4. J. S. Smith et al., J Clin Oncol 18, 636 (2000).-   5. G. Cairncross, R. Jenkins, Cancer J 14, 352 (2008).-   6. R. B. Jenkins et al., Cancer Res 66, 9852 (2006).-   7. C. A. Griffin et al., J Neuropathol Exp Neurol 65, 988 (2006).-   8. T. D. Bourne, D. Schiff, Nat Rev Neurol 6, 695.-   9. A. G. Knudson, Jr., Cancer 35, 1022 (1975).-   10. Materials and methods are in Example 5.-   11. L. Sastre, Clin Transl Oncol 13, 301.-   12. R. Xi, T. M. Kim, P. J. Park, Brief Funct Genomics 9, 405.-   13. S. H. Bigner et al., Am J Pathol 155, 375 (1999).-   14. Y. Jiao et al., Science 331, 1199 (2011).-   15. S. Jones et al., Science 330, 228.-   16. D. W. Parsons et al., Science 321, 1807 (2008).-   17. Y. Samuels et al., Science 304, 554 (2004).-   18. P. K. Vogt, A. G. Bader. S. Kang, Virology 344, 131 (2006).-   19. L. C. Cantley, Science 296, 1655 (2002).-   20. Y. Samuels, T. Waldman, Curr Top Microbiol Immunol 347, 21.-   21. Cancer Genome Atlas Research Network, Nature 455, 1061 (2008).-   22. X. S. Puente et al., Nature.-   23. H. Yan et al., N Engl J Med 360, 765 (2009).-   24. H. Yan, D. D. Bigner, V. Velculescu, D. W. Parsons, Cancer Res    69, 9157 (2009).-   25. L. Dang et al., Nature 465, 966.-   26. A. G. Knudson, J. Cancer Res. Clin. Oncol. 122, 135 (1996).-   27. S. J. Baker et al., Science 244, 217 (1989).-   28. G. Parmigiani et al., Genomics in press, (2008).-   29. G. Jimenez, A. Guichet, A. Ephrussi, J. Casanova, Genes Dev 14,    224 (2000).-   30. A. Garcia-Bellido. J. F. de Celis, Annu Rev Genet 26, 277    (1992).-   31. F. Roch, G. Jimenez, J. Casanova, Development 129, 993 (2002).-   32. L. Ajuria et al., Development 138, 915.-   33. S. Astigarraga et al., Embo J 26, 668 (2007).-   34. C. J. Lee, W. I. Chan, P. J. Scotting, J Neurooncol 73, 101    (2005).-   35. J. V. Olsen et al., Cell 127, 635 (2006).-   36. A. J. Wong et al., Proc Natl Acad Sci USA 89, 2965 (1992).-   37. S. H. Bigner et al., J Neuropathol Exp Neurol 47, 191 (1988).-   38. I. Vivanco, I. K. Mellinghoff, Curr Opin Oncol 22, 573.-   39. R. Duncan et al., Genes Dev 8, 465 (1994).-   40. H. H. Hsiao et al., Biochemistry 49, 4620 (2010).-   41. http://www.sanger.ac.uk/perl/genetics/CGP/cosmic.-   42. M. Kawamura-Saito et al., Hum Mol Genet 15, 2125 (2006).-   43. J. Grimwood et al., Nature 428, 529 (2004).

The invention claimed is:
 1. A method of identifying and treating anoligodendroglioma in a subject, comprising: detecting in a sampleobtained from the subject a mutation in CIC, FUBP1, or both CIC andFUBP1, wherein the mutation is selected from the group consisting of:CIC (R201W), CIC (R213P), CIC (R215Q), CIC (QK1517RD), CIC (A253T), CIC(P1485L), CIC (A306T), CIC (R215W), FUBP1 (E445X), FUBP1 (E411X), andcombinations thereof, wherein the sample comprises a brain tissuesuspected of being a brain tumor, or in cells or nucleic acids shed fromthe brain tumor, identifying the subject having the mutation as havingthe oligodendroglioma, and administering an anti-tumor agent to thesubject identified as having the oligodendroglioma.
 2. A method ofstratifying and treating a subject with a brain tumor, comprising:detecting in a sample obtained from the subject a mutation of CIC,wherein the mutation is selected from the group consisting of: CIC(R201W), CIC (R213P), CIC (R215Q), CIC (QK1517RD), CIC (A253T), CIC(P1485L), CIC (A306T), CIC (R215W), and combinations thereof, whereinthe sample comprises brain tumor tissue, or cells or nucleic acids shedfrom the brain tumor tissue, stratifying the subject having the mutationby assigning the subject to a group having an oligodendroglioma that isrefractory to EGFR inhibitors, and administering an anti-tumor agent tothe stratified subject having the oligodendroglioma that is refractoryto EGFR inhibitors, wherein the agent is not an EGFR inhibitor.
 3. Themethod of claim 2 further comprising: assigning the subject stratifiedas having the oligodendroglioma that is refractory to EGFR inhibitorswith the to a clinical trial group, wherein the clinical trial groupconsists of subjects whose brain tumors have the mutation.
 4. The methodof claim 1, wherein detecting in a sample is performed by sequencing anucleic acid in the sample.
 5. The method of claim 2, wherein detectingin a sample is performed by sequencing a nucleic acid in the sample. 6.The method of claim 1, wherein the mutation is in CIC, and theanti-tumor agent is not an EGFR inhibitor.